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1 AN INTERNATIONAL QUARTERLY JOURNAL OF BIOLOGY & LIFE SCIENCES B I O L I F E 2(2): ISSN (online): R E S E A R C H A R T I C L E ISOLATION AND IDENTIFICATION OF BIOLARVICIDE FROM SOURSOP (ANNONA MURICATA LINN.) AQUEOUS LEAF EXTRACT TO MOSQUITO (AEDES AEGYPTI LINN.) LARVAE Maghdu Nainamohamed Abubacker 1 *, Thiagarajan Deepalakshmi 2 and Chandran Sathya 3 1 PG & Research Department of Biotechnology, National College, Tiruchirappalli, India 2 PG & Research Department of Botany, National College, Tiruchirappalli, India 3 Department of Botany, Seethalakshmi Ramasamy College, Tiruchirappalli, India abubacker_nct@yahoo.com ABSTRACT To assess the biolarvicidal compound and larvicidal potentials of methyl ester of hexadeconic acid isolated from Annona muricata Linn. (A. muricata) against mosquito larvae Aedes aegypti Linn. (A. aegypti). The biolarvicidal compound methyl ester of hexadeconic acid was determined using GC-MS, while the larvicidal bioassay was carried out using different concentration of aqueous leaf extract of A. muricata against the larvae of A. aegypti in accordance with the standard protocol. Isolation and identification of larvicide bioactive compound from soursop (A. muricata) leaves against the larvae of A. aegypti mosquito, the transmitters of dengue fever has been carried out. The aquatic (rain water) extract of soursop leaves was an active larvicide agent with a lethal concentration (LC 50) varies with the concentration percentage and time. LC 50 for 2.5% = 3.0 h, 2.0% = 3.30 h, 1.5% = 3.15 h, 1.0% = 4.0 h and 0.5% = 5.0 h respectively. Total mortality occurred at 6.0 h for 2.5% concentration, 6.30 h for 2.0%, 7.30 h for both 1.5 and 1.0%, 8.0 h for 0.5% concentration. Removal of exoskeleton (dechitinising property) occurred between 1.0 h to 2.0 h for these concentrations tested for this work. Separation by TLC, analysis and identification with GC-MS showed peaks of 14 compounds, where there three dominant compounds with a retention time relatively close and have the abundance percentage that are large enough, which are identified as methyl ester of hexadecanoic acid, methyl ester of 9-octadecenoic acid (z), and 5-methyl-2-hexanone oxime with the most dominant compound which is methyl ester of hexadecamic acid which has a 44.11% abundance. The results justify the larvicidal potentials of bioactive compound from the leaf aqueous extract of A. muricata and the need to incorporate them in vector management and control. Key words : Soursop, biolarvicide, Annona muricata, Aedes aegypti, mortality. INTRODUCTION Mosquitoes are responsible for more diseases than any other group of Arthropods. To control mosquito population various pesticides are being used widely. Recent reports state that mosquitoes have become genetically and physiologically resistant to many conventional insecticides [1]. These factors have created the need for environmentally safe, biodegradable and target specific insecticides against mosquitoes. The search for such compounds has been directed extensively to the plant kingdom [2,3]. A. aegypti mosquito is a vector of several serious diseases to human, such as malaria, encephalitis, yellow fever, dengue fever, dengue hemorrhagic 579 Biolife 2014 Vol 2 Issue 1

2 fever, filariasis and arbovirus [4]. Dengue Hemorrhagic Fever (DHF) has no medicine or vaccine yet [5]. One way of preventing the spread of DHF is by prevention of dengue virus infection, by controlling and eradicating the vector to break off the disease transmissions [6]. Methods developed by WHO to combat dengue fever is the same as the method used to combat malaria, which is to eradicate the source of transmission, i.e. mosquito larvae [7]. Eradication of larvae is the key strategy of vector control programmes around the world [8]. A. aegypti mosquitoes lay eggs in clear water that is not directly affected by land and prefers containers indoor rather than outdoor due to the indoor temperature is relatively stable. A mosquito can lay eggs 4-5 times during her life with an average number of eggs ranges from eggs in a single spawn. Thus the total number of eggs produced by a single female mosquito is between eggs [9]. Physical control is conducted by managing the environment to prevent mosquito from breeding. Biological control is performed using predators and pathogenic organisms, while chemical control is carried out by applying synthetic insecticides to kill mosquitoes. Genetic control is done by spreading the sterile males into the ecosystem, and integrates control is performed by combining the various existing control techniques [10]. The most widely used mosquito control is the chemical control. The reason for this selection is the prompt results of this control. However, chemical control using synthetic insecticides actually causes adverse side effects, such as the mosquitoes could become resistant, human and livestock poisoning, contamination of vegetables and fruit gardens, as well as environmental pollution [11]. The development of new insecticides that are more environmentally friendly and do not pose hazard needs to be done. The use of bioinsecticides looks promising. Bioinsecticide or biological insecticide is an insecticide which is derived from plant material containing chemicals (bioactive) that are toxic to insects but are easily biodegradable in nature. So, it will not pollute the environment and relatively safe for human. Besides, vegetable insecticides are also selective [12]. Research on bioactive compounds in the Annonaceae family is growing rapidly. Acetogenin compounds from Annonaceae type were reported to have toxicity that is effective against insects of several orders such as Lepidoptera, Coleoptera, Homoptera and Diptera [13,14]. Other studies reported that Annonaceae family contains acetogenin that are larvicidal. Acetogenin also acts as an insecticide, acaricide, antiparasitic and bactericidal [15,16]. A. muricata Linn. (Soursop) seed extract contain annonacin, bullatacin, annonin VI, goniothalamin and sylvaticum act as insecticides [17,18]. Preliminary test results indicated that the ethanol extract of soursop seeds is an active agent of larvicide. Phytochemical test shows that ethanol soursop seed extract contains secondary metabolites compounds group of saponin, alkaloids and triterpenoids. These compounds are defense chemical compounds of plant produced in the plant tissue. They are toxic and can also act as the stomach and respiratory poison [19]. The present study will discuss the work in isolating and identifying the bioactive agents of larvicide from the soursop leaf aqueous extract against dengue fever mosquito (A. aegypti) larvae as the bioindicator. MATERIALS AND METHODS Collection and identification of plant material: Fresh leaves of A. muricata Linn. were collected from a private garden in Tiruchirappalli, Tamil Nadu (Figure a). The taxonomic identities of this plant was confirmed by flora of the Presidency of Madras [20]. The leaf material was washed under running tap water, air dried in shade and then homogenized to fine powder and stored in sterile air tight bottles for the experimental work. Isolation and identification of bioactive compound - Thin Layer Chromatography (TLC): Glass plates (4 cm 12 cm) were used in which 30 gm of silica gel mixed with 60 ml distilled 580 Biolife 2014 Vol 2 Issue 2

3 water slurry was prepared and coated on the glass plate to 0.25 cm thickness and dried for an hour at 110 C in an oven [21]. Preparation of leaf extract for bioactive compound: The dry powdered leaves (500 mg) of A. muricata was mixed with 5.0 ml of chloroform and ground into a paste, dried at room temperature. 1 ml of chloroform was added to the dried samples and spotted on the TLC plates. The TLC plates were kept in several eluent mixture with different polarities to separate the bioactive chemical compounds in chloroform extract has been tried. The eluents used were chloroform : n-hexane (8:2), chloroform : ethyl acetate (8:2), chloroform : acetone (8:2), n- hexane : acetone (9:1), and chloroform : acetone (9:1). Sample spotting on the TLC plate was done by using a micropipette in which the dot diameter 0.5 mm. The chloroform : acetone (9:1) was the best eluent since it was able to separate the four compounds contained in leaf extract [11]. Gas chromatography and mass spectroscopy (GC-MS) analysis: GC-MS analyses were performed using a GC Clarus 500 Perkin Elmer equipment equipped with a flame ionization detector and injector MS transfer line temperature of 230 C, fused silica capillary column Elite-5 MS (5% Diphenyl / 95% Dimethyl polysiloxane), m df, film thickness, carrier gas Helium at a flow rate of 28 cm/sec was used. 1 ml of extract mixed with methanol (80%) at a split rate of 10:1 was injected. The compound identification was accomplished by comparing the GC relative retention and mass spectra to those of authentic substances analysed under the same conditions, by their Retention Time (RT) and by comparison to reference compounds (Table 2). Preparation of leaf extract for biological activity: 500 mg of dried leaf powder was mixed with 100 ml of rain water constitute 0.5% concentration, while 1.0 g, 1.5 g, 2.0 g and 2.5 g constitute 1.0, 1.5, 2.0 and 2.5% concentration of leaf extract was used for the experimental work. The control organisms were maintained in rain water (Figure b). Toxicity Test: The various concentration of A. muricata leaf extract were tested for its biological activity against A. aegypti larvae in petridish. Each concentrations (20 ml) contained 20 larvae including control. Observations were carried out for 24 hours on the death of the larvae. The LC 50 and total mortality for each concentrations were recorded.. RESULTS The larvicidal effect of A. muricata leaf aqueous (rain water) extract of LC 50 and total mortality in terms of time was presented in Table-1. Table-1. Larvicidal effect of soursop (A. muricata Linn) leaf aqueous extract to mosquito (A. aegypti) larva CONSTITUENTS Concentration (%) No. of larvae tested Time (h) LC50 No. of larvae alive No. of larvae death Time (h) Total Mortality Rainwater (Control) Leaf extract in rain water Biolife 2014 Vol 2 Issue 2

4 The maximum concentration of 2.5% tested in this work shows LC 50 at 3.0 h, 2.0% at 3.30 h, 1.5% at 3.15 h, 1.0% at 4.0 h, 0.5% at 5.0 h (Figure-2d-h), the control organisms alive at 24.0 h and thereafter for all the developmental stages in subsequent days (Figures 1b, c). It is to be noted that in all the concentrations there was 100% mortality occurred with respect to time 6.0 h to 8.0 h (Figures-1j,k). Dechitinising property of larva was observed at 1.0 h (Figure-1i). DISCUSSION Larvicidal (Mosquito) efficacies was reported by many workers [22-26]. In all these reports organic solvents were used in the plant extract. In this report the natural habitat of A. aegypti rain water source alone tested with A. muricata leaf extract. The GC-MS analysis revealed the presence of major bioactive compound methyl ester of hexadoconic acid (Figure 2). Figure-1. Larvicidal effect of soursop (Annona muricata Linn) leaf aqueous extract to mosquito (Aedes aegypti) larva (a-annona muricata Linn. Twig, b-aedes aegypti larvae in rain water, c-control (rain water), d-a. muricata aqueous (rain water) leaf extract 0.5%, e-concentration 1.0%, f-1.5%, g-2.0%, h-2.5, i- Dechitinising property of larva (1 hour), j-morphology of dead larva (3 hours) and k-morphology of dead larva (6 hours) 582 Biolife 2014 Vol 2 Issue 2

5 Table-2. Bioactive compounds identified in Annona muricata leaf extract No. RT Name of the Compound Molecular formula MW Peak Area % Hexanal, O-methyloxime C 7 H 15 NO Butanal, O-methyloxime C 5 H 11 NO Propanone,oxime C 3 H 7 NO Methyl-2-hexonone oxime C 7 H 15 NO ,2-Ethanediamine, N-[2-aminoethyl) C 4 H 13 N Butanamide, 4-cyano-N-methyl C 6 H 10 N 2 O ,13-Tridecanediol, diacetate C 17 H 32 O Hexadecanoic acid, methyl ester C 17 H 34 O Octadecenoic acid (Z)-,methyl ester C 19 H 36 O Undecen-1-yl acetate C 13 H 24 O Acetamide, N-acetyl-N-methyl C 5 H 9 NO Cyclohepten-1-one C 7 H 10 O Hexen-2-one, O-methyloxime C 7 H 13 NO Acetic acid, 2-methylpropyl ester C 6 H 12 O Figure-2. GC-MS Chromatogram of Annona sp. The total mortality of A. aegypti larvae between 6.0 to 8.0 h might be due to this bioactive compound. The present work will help to incorporate this bioactive compound in vector management and control. ACKNOWLEDGEMENTS Author (MNA) wish to thank DST-FIST, Government of India, New Delhi for providing the infrastructure facilities to the Department of Botany, National College, Tiruchirappalli, Tamil Nadu. Authors also expresses thanks to Padmavibhushan Dr. V. Krishnamurthy, President, Sri. K. Ragunathan, Secretary and Dr. K. Anbarasu, Principal, National College, Tiruchirappalli for all the supports and encouragement given to PG and Research Department of Biotechnology to carry over the research work. 583 Biolife 2014 Vol 2 Issue 2

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